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PubMed1991

Quantitative isolation of RNA from human platelets.

We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia.

Blood Platelets↗
PubMed1982

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth.

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.

Acridine Orange↗
PubMed2004

Isolation of high quality RNA from Streptomyces.

Several molecular techniques require high quality RNA, completely free of DNA. Standard methods to isolate total RNA from Streptomyces spp. are based on the application of a 'Modified Kirby Mix' [Practical Streptomyces Genetics. The John Innes Foundation, Norwich, pp. 613]. Here we present an alternative procedure using Triton X-100 and EDTA for the isolation of total RNA from Streptomyces.

Edetic Acid↗
PubMed1991

[The content of DNA, RNA and total protein in different skeletal muscles, in the diaphragm, in the right and left heart ventricles and in the musculature of the esophagus, the stomach and the urinary bladder of normally developed piglets and splayleg piglets of different body weights].

Studies were conducted into concentrations of RNA, DNA, and total protein in various skeletal muscles, diaphragm, right and left ventricles as well as in musculature of the oesophagus, stomach, and urinary bladder of 25 normally developed piglets and 24 sprayleg piglets of differentiated body weight (B.W.). DNA concentration was found to be at its highest in piglets of lowest B.W. The Protein: DNA-quotient increased along with growing B.W. The average value of this quotient in the anterior tibial muscle and brachial triceps muscle of sprayleg piglets was lower than that in normally developed piglets. Average values of the RNA:DNA quotients in the brachial triceps muscle, anterior section of the long dorsal muscle, left ventricle, and stomach muscles were also lower in sprayleg piglets. In newborn piglets, DNA concentrations in ventricles as well as in the muscles of the stomach and oesophagus were higher than those recordable from skeletal muscles. Causes of incapability of newborn piglets to stand on their own feet are discussed in some detail.

Animals↗
PubMed1983

Hormonal regulation of transcription of rDNA. Inhibition of transcription during glucocorticoid-mediated inhibition of proliferation of lymphosarcoma P1798 cells in culture.

A lymphosarcoma cell line designated P1798.S6M has been characterized with respect to glucocorticoid responsiveness in culture. Cells ceased to proliferate in the presence of 10(-7) M dexamethasone. Cell viability remained high and glucocorticoid-sensitive cells could be rescued from cultures treated with dexamethasone for 24 or 48 h. These data indicate that P1798.S6M undergoes reversible arrest in the presence of dexamethasone. The system was used to study the effects of mitotic arrest upon transcription of rDNA. Incorporation of [methyl-3H]methionine into rRNA was rapidly inhibited and pulse-chase experiments indicated that 28 S RNA was not synthesized after 24 h of exposure to dexamethasone. Hybridization studies indicated that the amount of pre-rRNA was reduced by 90 to 95% in cells treated for 24 h. Transcription studies were carried out in isolated nuclei. Twenty-four hours after addition of dexamethasone, template-bound RNA polymerase I activity decreased by 89 to 96%. Total RNA polymerase I activity did not decrease, whereas disengaged nuclear polymerase I activity increased dramatically. Filter hybridization studies indicated that labeling of nascent pre-rRNA chains in vitro was inhibited 93%. These data are interpreted as follows: Dexamethasone reversibly inhibits proliferation of P1798.S6M cells and transcription of rDNA. Total RNA polymerase I activity does not decrease, but the amount of template-bound enzyme is reduced with a concomitant increase in the amount of disengaged polymerase I. This indicates that initiation of transcription is inhibited in cells undergoing mitotic arrest in the presence of dexamethasone.

Animals↗
PubMed1969

Changes in ribonucleic acid fractions during maturation of mimosa epicotyl tissues.

Total RNA and DNA of mimosa epicotyl tissues were extracted and the RNA fractionated into specific soluble RNAs (sRNAs) at different times during late germination. Epicotyls collected at each time contained qualitatively comparable meristematic and developing tissues, while mature tissues increased. Quantitative ratios of total RNA to DNA and total sRNAs to approximated ribosomal RNA (rRNA) varied consistently during development. Terminal nucleosides of sRNA did not vary in any consistent pattern through development. On the other hand, regular changes in quantitative ratios of specific sRNA groups were observed during development.

DNA↗
PubMed1979

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.

Gamma Rays↗
PubMed1993

Changes in levels of mRNA encoding myosin heavy chain in porcine trachealis during ontogenesis.

We determined the steady-state level of mRNA for myosin heavy chain (MHC) from airway smooth muscle during maturation in domestic swine. Tissues were excised, and airway smooth muscle was dissected from three neonatal (NEO), three 2-wk-old swine (2ws), three 10-wk-old swine (10ws), and three adult swine. Total RNA was isolated, fractionated, and transferred to a nitrocellulose membrane (Northern blot). A single-stranded oligonucleotide of 63-nt was synthesized corresponding to the 3' coding region of the chicken gizzard MHC cDNA. This region appeared to be highly conserved (92% nucleotide sequence homology with the corresponding portion of rabbit uterine smooth muscle MHC cDNA). Northern blots, which were loaded with equivalent quantities of total RNA, were probed with gamma 32P-labeled synthetic oligonucleotide, and, under stringent washing conditions, the 5' end-labeled DNA was hybridized to a single band of the expected molecular weight. The mRNA for total myosin was quantified using autoradiograms of blots, and signal intensity was measured as integrated areas expressed as arbitrary densitometric units x mm (AU). The content of mRNA for MHC was substantially greater in NEO than in more mature animals; maximal area was 1.33 +/- 0.15 AU for NEO, 0.33 +/- 0.05 AU for 2ws, 0.30 +/- 0.04 AU for 10ws, and 0.34 +/- 0.08 AU for adult swine (P < 0.05, NEO versus 2ws, 10ws, and adult). Rehybridization of each blot with a 28S ribosomal RNA probe confirmed comparable total RNA loadings for all tissue samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1998

Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay.

End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.

Adolescent↗
PubMed1989

Possible involvement of inhibin in altered follicle-stimulating hormone (FSH) secretion during dissociated luteinizing hormone (LH) and FSH release: unilateral castration and experimental cryptorchidism.

Male rats were either unilaterally or bilaterally castrated, or were rendered cryptorchid when they were either 15 or 45 days old. Subsequently, blood was sampled over the next several weeks and plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and immunoreactive inhibin-alpha (irI alpha) levels were measured by specific radioimmunoassays (RIAs). At the end of the experiment, gonadal expression of inhibin-alpha, inhibin-beta A, and inhibin-beta B subunits was measured by S1 nuclease analysis and in situ hybridization. In both age groups, bilateral castration (BC) produced the expected marked (p less than or equal to 0.01) increases in plasma LH and FSH levels, and concomitant decreases in T and irI alpha secretion within 1 - 2 days after surgery. In 15-day-old animals, unilateral castration (UC) significantly increased FSH and decreased circulating levels of irI alpha, but did not measurably alter LH or androgen production. At 7 days after surgery, the level of inhibin mRNA in the remaining testis was unchanged. In 45-day-old animals, UC caused a measurable increase in FSH, with little or no changes in the circulating levels of irI alpha. Plasma T levels were lowered (p less than or equal to 0.05) by UC; however, there were no statistical changes in LH levels in these UC rats. Finally, T administration markedly reversed UC-induced increase in FSH secretion in both age groups. Androgen therapy also interfered with inhibin release in 45-day-old, but not in 15-day-old rats. In rats 15 days old at the time of surgery, cryptorchidism produced a small but measurable increase (p less than or equal to 0.05) in LH release at Week 6 only, which was accompanied by a significant (p less than or equal to 0.01) decline in T secretion. Plasma FSH levels were elevated at all times in cryptorchid rats, and at 2, 4, and 6 wk, these levels were not statistically distinguishable (p greater than 0.05) from those of castrated animals. In this group of rats, cryptorchidism caused a transient increase (p less than or equal to 0.05) in irI alpha values 1 wk after surgery, but no changes at later times. Finally, measurement of testicular inhibin-alpha subunit messenger RNA (mRNA) levels showed an approximately 2-fold increase compared to total RNA levels in the testis. However, because of the significant decrease in total RNA levels per testis caused by cryptorchidism, the absolute change in inhibin-alpha subunit mRNA levels per testis corresponded to an approximately 3-fold decrease.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Discoordinate gene expression of aggrecan and type II collagen in experimental osteoarthritis.

OBJECTIVE: To quantify the gene expression of aggrecan core protein and type II collagen in an experimental animal model of osteoarthritis (OA). METHODS: Total RNA was extracted from the articular cartilage of unoperated knee joints and from OA joints produced by anterior cruciate ligament transection. The relative amounts of type II collagen and aggrecan core protein messenger RNA (mRNA) were evaluated by Northern blot analysis. RESULTS: Total RNA was elevated 2.5 times, aggrecan mRNA was elevated 2 times, and type II collagen mRNA was elevated 8 times, in OA knees compared to unoperated controls. CONCLUSION: Chondrocytes are activated metabolically in response to joint injury. Discoordinate gene expression of aggrecan and type II collagen is characteristic of early experimental OA, and we speculate that it may contribute to its pathogenesis.

Aggrecans↗
PubMed2005

Distribution of gonadotropin-releasing hormone (GnRH) by in situ hybridization in the tunicate Ciona intestinalis.

Gonadotropin releasing hormone (GnRH) is the key hypothalamic neurohormone that is critical in its role of reproduction in all vertebrates. There are currently twenty-four known forms of GnRH that have been identified, 14 in vertebrates and 10 in invertebrates. In tunicates, the primary structure of nine forms have been identified, all of which have been shown to stimulate gamete release. However, the distribution and function of the various GnRH peptides in tunicates have not been fully examined. Therefore, the objective of this study was to determine tissue specific expression of Ci-gnrh-1 and Ci-gnrh-2 in an adult tunicate, Ciona intestinalis, using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. To examine the expression of the two GnRH genes, total RNA and genomic DNA were isolated from whole animals. Total RNA from neural tissue (cerebral ganglion and neural gland), testis, ovary, heart, and hepatic organ were also isolated. Results from RT-PCR indicated both forms are only expressed in the neural tissue. We extended these studies using fluorescent dual label in situ hybridization. GnRH expression was confirmed to be in the cerebral ganglion bordering the neural gland. These current data along with previous studies suggest that GnRH may be involved in reproduction in the protochordate.

Animals↗
PubMed1999

Recovery of high-integrity mRNA from brains of rats killed by high-energy focused microwave irradiation.

Following decapitation, during the brief time period of postmortem brain tissue collection, significant changes in neuro-metabolite levels can occur. To circumvent such changes, we routinely kill rats using 1 to 2 sec pulses of focused high-energy microwave irradiation (10 kW). The effects of high-energy microwave irradiation on total RNA and mRNA integrity in brain however, are unknown. Total RNA recovery, per gram wet weight, in brain regions of microwaved rats was less than 50% of that in rats killed by decapitation. Formaldehyde agarose gel electrophoresis showed that ribosomal RNA components were highly degraded in all brain regions of microwaved rats. In contrast, poly A+mRNA, as measured by poly A+driven cDNA synthesis and Northern analysis, in brain samples of microwaved or decapitated rats was of equal integrity and quantity when expressed per mg tissue weight. Furthermore, positive RT-PCR products for GAPDH and TNF-alpha were observed in brain regions of both microwaved and decapitated rats. These observations indicated that high-energy focused microwave irradiation does not reduce mRNA abundance and integrity. Thus, this method of animal sacrifice can be used to simultaneously study, accurately and precisely, levels of brain metabolites as well as molecular biological events in discrete brain regions of experimental animals without postmortem interference.

Adenosine↗
PubMed1997

Inflammatory cytokine expression is correlated with the level of human immunodeficiency virus (HIV) transcripts in HIV-infected placental trophoblastic cells.

The inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) have been associated with increased human immunodeficiency virus (HIV) expression and enhanced lymphocyte adhesion to trophoblastic cells in experimental systems. To determine if there is a correlation between the expression of these cytokines and the levels of HIV transcripts in trophoblasts of term placentas from HIV-infected women, we studied the placentae of 30 HIV-positive and 13 control gravidae. Twenty-three of the HIV-positive women received zidovudine (ZDV) as prophylaxis against HIV vertical transmission; only one of the seven women who did not receive ZDV was a transmitter, for an overall vertical transmission rate of 3.8%. Cytokine production was measured by enzyme-linked immunosorbent assay in the supernatants of trophoblastic cell cultures. Additionally, cytokine transcripts and HIV gag sequences were determined by a quantitative reverse transcription-PCR assay. In general, trophoblastic cells of HIV-positive placentas expressed significantly higher levels of IL-1beta, IL-6, and TNF-alpha than those of control placentas. All placentas from HIV-positive women expressed HIV gag transcripts at either a low (<156 copies per microg of total RNA) or a high (>156 copies per microg of total RNA) level. There was a statistically significant positive association between the basal level of TNF-alpha production and the level of HIV gag transcripts of HIV-positive placental trophoblastic cells. Nevertheless, these data, coupled with a low transmission rate, would indicate that some other factors, perhaps working in concert with cytokines, are necessary for vertical transmission of HIV from mother to infant.

Cells, Cultured↗
PubMed1995

The identification of bacterial gene expression differences using mRNA-based isothermal subtractive hybridization.

We describe a method for isolating and determining differences in gene expression between related bacterial strains. The method is based upon differences in mRNA expression. To demonstrate this procedure, cDNA generated from total RNA of Listeria monocytogenes serotype 1/2a was hybridized to total RNA from a Tn916 mutant of serogroup 1/2a (M3) that was deficient in the production of listeriolysin O, the product of the hly gene. The single-stranded cDNA fragments remaining after hybridization represent the difference in expressed genes between the two strains. These subtraction products were used as hybridization probes to identify the corresponding hly gene in a Southern hybridization.

Base Sequence↗
PubMed2005

[Cloning and characterization of a novel mouse aging-related gene Arzc].

OBJECTIVE: To clone and identify novel mouse aging-related genes. METHODS: The improved differential display reverse transcription-polymerase chain reaction method was used to analyze the differential expression of old and young BALB/C mouse cortex tissues. The novel expression sequence targets (EST) with differential expression were further confirmed by Northern blot and used as original sequence to clone the full-length cDNA sequence by bioinformatics technique. The reverse transcription products of BALB/C mouse cortex total RNA was used as the amplification template. RESULTS: A total of 42 differentially expressed EST were obtained. Homologic analysis showed that 29 EST represented known cDNAs and 13 were probably new EST. One of the EST, which were confirmed to have down regulative expression in the old mouse cortex tissues by Northern blot analysis, was used to obtain the full-length cDNA sequences from reverse transcription products of total RNA of BALB/C mice cortex by bioinformation method. A 1 382 bp of cDNA sequence including the EST was obtained and designed as Arzc (GenBank accession number: AY344585). The Arzc cDNA contains an open reading frame of 261 bp and encodes a peptide of 86 amino acids residuals. FASTA analysis showed that the deduced peptide had a homology with a portion of integrase/recombinase, a member of a phage integrase family. CONCLUSION: A novel mouse gene that is probably related to aging was identified and cloned.

Aging↗
PubMedDate not supplied

Enterotrophic effect of insulin-like growth factor-I but not growth hormone and localized expression of insulin-like growth factor-I, insulin-like growth factor binding protein-3 and -5 mRNAs in jejunum of parenterally fed rats.

BACKGROUND: Administration of insulin-like growth factor (IGF)-I, but not growth hormone (GH), stimulates mucosal hyperplasia in surgically stressed rats with intestinal atrophy induced by hypocaloric total parenteral nutrition (TPN). Our aim was to characterize the basis for this disparity in enterotrophic action by assessing the relationships between stimulation of intestinal growth, nutritional adequacy, and localization of expression of IGF-I, insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mRNAs in jejunum. METHODS: Rats were maintained with TPN for 8 days and treated with IGF-I or GH and adequate nutrition for 5 days after recovery from surgery. Jejunal mass, morphology, and sucrase activity were assessed. Localization of expression of IGF-I, IGFBP-3, and IGFBP-5 mRNAs in jejunum was accomplished by in situ hybridization. RESULTS: Serum IGF-I and body weight gain were significantly increased by IGF-I or GH. Jejunal mucosal dry mass, morphology, and sucrase activity were improved with IGF-I but not GH. There were no differences in IGF-I mRNA. IGFBP-3 mRNA was localized in the lamina propria of the villi. IGF-I or GH stimulated IGFBP-3 expression. IGF-I strongly stimulated IGFBP-5 expression in the lamina propria and the muscularis and induced a twofold increase in IGFBP-5 mRNA based on RNase protection assay of intact jejunum total RNA. GH induced a modest increase in IGFBP-5 expression in the muscularis with no effect on intact jejunum total RNA. CONCLUSIONS: The GH resistance observed in the jejunal mucosa of TPN rats cannot be fully explained by inadequate nutrition. The expression of IGFBP-5 in the lamina propria suggests it may modulate the enterotrophic action of exogeneous IGF-I.

Animals↗
PubMed2002

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.

Autolysis↗